Conversion of the insoluble cellulose and hemicellulose polymers found in plant biomass into soluble sugars represents a major bottleneck for the biofuel industry. In order to accomplish this conversion, chemical pretreatment and enzyme hydrolysis are usually required.
Among chemical pretreatments, ammonia fiber expansion (AFEX) alkaline pretreatment has many advantages, such as (1) being a dry to dry process that does not produce a wash or liquid stream, (2) producing a chemically similar biomass composition after pretreatment, and (3) resulting in cleaved lignin-carbohydrate complexes without physically extracting hemicelluloses or lignin into separate process streams. Because alkaline pretreatments retain all constituent fractions of biomass, these pretreatments must be paired with the use of a complex mixture of cellulases and hemicellulases to achieve effective hydrolysis. It would be desirable to decrease the number of enzymes required to provide high yield conversion of polysaccharides present in AFEX-pretreated biomass.
Furthermore, a new variant chemical pretreatment called extractive AFEX (E-AFEX) leads to the production of cellulose III, an unnatural form of cellulose (Chundawat, et al., 2011). The extractive AFEX process not only generates cellulose III as a potentially more easily hydrolyzed allomorph, but also partially removes lignin. Both of these beneficial effects should promote enzymatic processing. Enzyme cocktails which hydrolyze cellulose III may have unique complementarity and thus corresponding utility when combined with the E-AFEX process.
In nature, the complex ultra-structure of the plant cell wall requires the participation of a mixture of many different enzymes to efficiently carry out the conversion of biopolymers into soluble sugars. This mixture is called an enzyme cocktail. An enzyme cocktail simultaneously provides cellulase, xylanase, mannanase, and enzymes having other catalytic activities. Cocktails of enzymes that have high hydrolyzing efficiency for either AFEX treated or extractive AFEX (E-AFEX) treated biomass will have great potential in the biofuel industry.
Chemical pretreatments and enzymes must be used together in order to achieve a high-yield deconstruction of biomass into fermentable sugars. As many as 18 different purified enzymes may be required to give a high yield conversion of AFEX pretreated biomass into soluble sugars, because AFEX pretreatment does not destroy the many types of bonds found in hemicellulose. Approaches that result in a reduced number of enzymes required for an efficient biomass hydrolysis would be beneficial to the biofuel industry, because it would be easier to manufacture a smaller number of enzymes, and the total weight of enzyme required may also be decreased.
One way to simplify the composition of enzyme cocktails is to use multifunctional enzymes, whereby a single multifunctional enzyme can replace two or more monofunctional enzymes in the cocktail. As a result of such substitutions, a less complex enzyme cocktail containing fewer enzymes may be used.
Accordingly, there is a need in the art for a multifunctional enzyme which can hydrolyze the three major materials of plant cell walls: cellulose, xylan, and mannan. Such a multifunctional enzyme could replace two or more enzymes in a conventional enzyme cocktail, while providing advantages in specific activity and stability of the cocktail.